To quantify fluorescence-labeled biomolecules, an analysis apparatus for irradiating the sample solution with photon in such a way that photons may converge at a point (hereinafter referred to as “focusing point”) within the solution to excite the fluorescence, collecting the fluorescence irradiated by the biomolecules in the solution and transmitting through the sample cell with the collection lens, and thus detecting only the photons forming image on a pinhole placed on the conjugate point of the focusing point and transmitting through the pinhole is generally used. By detecting only the photons transmitting through the pinhole, the volume for detecting the fluorescence in the sample (detection volume) is limited to the vicinity of the focusing point, and background light (B) such as luminescence of solution at anywhere other than the vicinity of the focusing point, scattering light at the surface of the sample cell is blocked, and thus it is possible to detect the fluorescence from biomolecules in the detection volume (signal, S) with a good signal to background (S/B) ratio.
The detection system of this type of apparatus is generally constituted as shown in FIG. 1. Excited beam is focused at the sample 1 in the sample cell 2, and the fluorescence 4 emitted from the focusing point 3 is collected at the pinhole on the pinhole plate 6 created at a conjugate position with the focusing point 3 by means of the collection lens 5, and is detected by the photodetector 7 passing through the pinhole. In reality, the lens 5 is a combination lens made of a plurality of lens, incorporating a filter, a dichroic mirror and the like between the lens. In FIG. 1 which is a schematic illustration showing the basic principle, however, we used a simplified structure for its illustration. As B is reduced to a sufficiently low level in a system like the one shown in FIG. 1, the signal to noise (S/N) ratio of fluorescence detection is proportionate to √S. Therefore, it is necessary to increase the value of S itself in order to increase the S/N ratio value. There are two means for increasing the value of S. One is to increase the intensity of photon being irradiated. And the other is to increase the efficiency of fluorescence collection. Any irradiation of light excessively strong risks to destroy the fluorescent substance labeling the biological specimen, and therefore there is a limit to this method. Accordingly, the efficiency of fluorescence collection of the detection system must be increased to the maximum extent possible. In other words, the numerical aperture (NA) of the detection system must be increased to the maximum extent possible. In order to detect the fluorescence from one molecule with a good S/N ratio, a NA larger than one is preferable. However, in order to achieve NA>1 by using a sample cell 2 having a generally flat bottom, the lens 5 must be immersed in a liquid as described in JP-A No. 85443/2004. In other words, the space between the lens 5 and the sample cell 2 must be filled with a liquid. As the liquid-immersion system involves a step of filling the space between the lens and the sample cell with a liquid, its operability is somewhat lower than that of the dry system. In case of analyzing a variety of samples while moving the sample cell, the troublesomeness of operation is remarkable. And this leads to a high risk of generating bubbles in the liquid with which the cell is filled and of impeding the transmission of fluorescence. Although there are systems wherein the process of charging liquid or removing bubbles is automatized as shown in JP-A No. 85443/2004, a rise in the cost of equipment is unavoidable, and the troublesomeness of filling liquid and replacing sample cells remains.
Although in some documents the focusing point in the sample and the conjugate point are called “focal point,” this is an imprecise expression, and in the present specification we used the term “focal point” only in the sense of “a point where parallel pencils introduced to the lens from the sample side are focused in the side opposite the sample side.” In order to clarify this point, in FIG. 1, we indicated fluorescence of virtual parallel pencils 4′ and focal point 8. Thus, according to the prior art, the focal point 8 is not located in the pinhole.
As shown in the prior art, it is possible to realize NA>1 even in a non-liquid immersion system (dry system) by creating a curved surface on the bottom of the sample cell. When this is combined with a detection system with a pinhole as shown in FIG. 1, a system shown in FIG. 2 can be created. Since the light coming out of the sample cell shown in the prior art is divergent light, the pinhole of the pinhole plate 6 is not located at the focusing point 8 of the lens 5 also in the system of FIG. 2. According to this system, it is possible to realize NA>1 in a dry system.